CN110129471A - A nucleic acid sequence for detecting rice plant ZUPM01 and its detection method - Google Patents

Abstract

The present invention relates to a nucleic acid sequence for detecting rice plant ZUPM01 and a detection method thereof. The nucleic acid sequence of the rice plant includes SEQ ID NO: 1 or its complementary sequence, or SEQ ID NO: 2 or its complementary sequence. The rice plant ZUPM01 of the present invention has good tolerance to glyphosate herbicide and low phosphorus stress, and the detection method can accurately and quickly identify whether a biological sample contains the DNA molecule of the transgenic rice event ZUPM01.

Description

A nucleic acid sequence for detecting rice plant ZUPM01 and its detection method

technical field

The invention relates to the field of plant biotechnology. Specifically, it relates to a nucleic acid sequence for detecting rice plant ZUPM01 and its detection method, in particular to a transgenic rice event ZUPM01 that is tolerant to glyphosate herbicide application and low phosphorus stress and is used for detecting whether The nucleic acid sequence comprising the specific transgenic rice event ZUPM01 and its detection method.

Background technique

Field weeds compete with crops for water, fertilizer, light and growth space, directly affecting crop yield and quality. At the same time, many weeds are intermediate hosts of crop pathogenic bacteria and pests, and are one of the important biological limiting factors for crop production. According to statistics from the Food and Agriculture Organization of the United Nations, the annual loss of food production due to weeds in the world is as high as 95 billion US dollars, which is equivalent to the loss of 380 million tons of wheat, which is more than half of the global wheat production in 2009. Of the $95 billion in economic losses, poor developing countries have borne about $70 billion (FAO. The lurking menace of weeds [J/OL].(http://www.fao.org/news/story/en /item/29402/icode/), 2009-08-11.). Therefore, effective control of field weeds is one of the important measures to promote grain production. In my country, there are more than 40 kinds of weeds that harm rice, and there are more than 10 kinds of weeds that are more harmful. Weeds can reduce rice yield by 10-20% in normal years, and even up to 30-50% in severe cases. In addition, with the acceleration of the migration of rural population to cities in my country, the scale and mechanization of rice planting is a foreseeable trend, which makes the traditional manual weeding method unrealistic. At present, the selective herbicides widely used in the market are applied in a large amount and have a long residual period, which easily affects the normal growth of the next crop. Bacterial herbicides such as glyphosate have the characteristics of high efficiency, low toxicity, easy degradation, and no residue. But they are not selective for weeding and cannot be used directly in the growing season of crops. Breeding rice resistant to such herbicides through transgenic technology can overcome this problem. Spraying 1-2 times during the rice growth period can effectively solve the weed problem, reducing the amount of herbicides and input costs. Therefore, herbicide-tolerant transgenic rice has very broad application value and market potential.

Phosphorus is an important component of nucleic acid, phospholipids and ATP in plants. As a substance for energy transfer in plants, it can activate proteins in the body and regulate the entire metabolic process of plants. Among the various inorganic nutrients needed by plants, phosphorus is one of the most important elements, but it is extremely difficult to obtain from the soil. Although phosphorus is abundant in ecosystems, the chemical form of phosphorus assimilated by plants is mainly inorganic phosphorus-phosphate (Pi). The distribution of phosphate in the soil is extremely uneven, and most of the phosphate cannot move freely, which is not conducive to the absorption of the root system (Raghothama K G. Phosphate Acquisition[J]. Annual Review of Plant Physiology and Plant Molecular Biology, 1999, 50 (1 ): 665-693.). Therefore, the supply of soil available phosphorus and the ability of plants to absorb phosphorus nutrients have become one of the main determinants of plant growth and development (Schachtman D P, Reid R J, and Ayling SM.Phosphorus Uptake by Plants: From Soil to Cell[J ]. Plant Physiol, 1998, 116(2): 447-53.).

As a non-renewable resource, the phosphate rock on the earth is gradually decreasing under the continuous exploitation and utilization of human beings. According to data from the US Geological Survey, the total amount of phosphate mining in the world in 2008 was 160 million tons, and the demand for chemical fertilizers will grow at an annual rate of 2.5%-3% in the next five years. If things go on like this, the world's phosphate rock resources can only support human needs for about 125 years (Gilbert N. Environment: The disappearing nutrient [J]. Nature, 2009, 461(7265): 716-8.). At the same time, excessive application of phosphorus will also cause great harm to the environment; therefore, improving the uptake and utilization of phosphorus by crops is of great significance to both ecology and agricultural economy.

The expression of exogenous genes in plants is known to be influenced by their chromosomal location, possibly due to the proximity of chromatin structure (such as heterochromatin) or transcriptional regulatory elements (such as enhancers) to the integration site. For this reason, it is usually necessary to screen a large number of events before it is possible to identify commercially viable events (ie, events in which the introduced target gene is optimally expressed). For example, it has been observed in plants and other organisms that the amount of expression of an introduced gene can vary considerably between events; there may also be differences in the spatial or temporal pattern of expression, such as the relative expression of the transgene between different plant tissues There are differences in which the actual expression pattern may not correspond to that expected based on the transcriptional regulatory elements in the introduced gene construct. Therefore, it is often necessary to generate hundreds to thousands of different events and to screen these events for a single event with the expected amount and pattern of transgene expression for commercialization purposes. Events with the expected amount and pattern of transgene expression can be used to introgress the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. The progeny produced by this crossing method maintained the transgene expression characteristics of the original transformant. Applying this pattern of strategies can ensure reliable gene expression in many varieties that are well adapted to local growing conditions.

It would be beneficial to be able to detect the presence of a particular event to determine whether the progeny of a sexual cross contain the gene of interest. In addition, methods to detect specific events would help to comply with regulations, such as the need for formal approval and labeling of food derived from recombinant crops before being placed on the market. Detection of the presence of the transgene is possible by any of the well-known polynucleotide detection methods, such as polymerase chain reaction (PCR) or DNA hybridization using polynucleotide probes. These assays usually focus on commonly used genetic elements such as promoters, terminators, marker genes, etc. Therefore, unless the sequence of the chromosomal DNA adjacent to the inserted transgenic DNA ("flanking DNA") is known, this approach cannot be used to distinguish between different events, especially those produced with the same DNA construct. event. Therefore, transgene-specific events are now commonly identified by PCR using a pair of primers spanning the junction of the inserted transgene and flanking DNA, specifically a first primer containing the flanking sequence and a second primer containing the inserted sequence.

Contents of the invention

The object of the present invention is to provide a nucleic acid sequence for detecting rice plant ZUPM01 and its detection method. The transgenic rice event ZUPM01 has good tolerance to glyphosate herbicide and low phosphorus stress, and the detection method can be accurate and fast To accurately identify whether a biological sample contains DNA molecules of a specific transgenic rice event ZUPM01.

To achieve the above object, the present invention provides a nucleic acid sequence comprising SEQ ID NO: 1 or its complementary sequence, and/or SEQ ID NO: 2 or its complementary sequence, the nucleic acid sequence is derived from transgenic rice Event ZUPM01.

Further, the nucleic acid sequence comprises SEQ ID NO: 3 or its complementary sequence, and/or SEQ ID NO: 4 or its complementary sequence.

Furthermore, the nucleic acid sequence includes SEQ ID NO: 5 or its complementary sequence.

Said SEQ ID NO: 1 or its complementary sequence is a sequence of 22 nucleotides in length near the insertion junction at the 5' end of the inserted sequence in the transgenic rice event ZUPM01, said SEQ ID NO: 1 or its complementary sequence The complementary sequence spans the genomic DNA sequence on the left flank of the rice insertion site and the DNA sequence at the 5' end of the left border of the insertion sequence, and the presence of the transgenic rice event ZUPM01 can be identified by including said SEQ ID NO: 1 or its complementary sequence . The SEQ ID NO: 2 or its complementary sequence is a sequence of 22 nucleotides in length near the insertion junction at the 3' end of the inserted sequence in the transgenic rice event ZUPM01, and the SEQ ID NO: 2 or its complementary sequence The presence of the transgenic rice event ZUPM01 can be identified by the DNA sequence spanning the 3' end of the right border of the insertion sequence and the right flank genomic DNA sequence of the rice insertion site, including said SEQ ID NO: 2 or its complementary sequence.

In the present invention, the nucleic acid sequence may be at least 11 or more continuous polynucleotides (first nucleic acid sequence) of any part of the transgene insertion sequence in the SEQ ID NO: 3 or its complementary sequence, or the At least 11 or more contiguous polynucleotides (second nucleic acid sequences) of any part of the 5' left flank rice genomic DNA region of SEQ ID NO: 3 or its complementary sequence. Said nucleic acid sequence may further be homologous or complementary to a portion of said SEQ ID NO:3 comprising the entirety of said SEQ ID NO:1. When a first nucleic acid sequence and a second nucleic acid sequence are used together, these nucleic acid sequences comprise a set of DNA primers in a DNA amplification method that produces an amplification product. When the amplified product generated in the DNA amplification method using the DNA primer pair is an amplified product comprising SEQ ID NO: 1, the presence of the transgenic rice event ZUPM01 or its progeny can be diagnosed. As is well known to those skilled in the art, the first and second nucleic acid sequences need not consist solely of DNA, but may also include RNA, a mixture of DNA and RNA, or DNA, RNA or other nucleosides that do not serve as templates for one or more polymerases Combinations of acids or their analogs. In addition, the probes or primers of the present invention should be at least about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, which can be selected from Nucleotides described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. When selected from the nucleotides shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, the probes and primers may be at least about 21 to about 50 or more contiguous Nucleotides. Said SEQ ID NO: 3 or its complementary sequence is a sequence of 732 nucleotides in length near the insertion junction at the 5' end of the inserted sequence in the transgenic rice event ZUPM01, said SEQ ID NO: 3 or its complementary sequence The complementary sequence consists of the rice left flank genomic DNA sequence (SEQ ID NO: 3 nucleotides 1-486) of 486 nucleotides, the left border DNA sequence of the pPHF1G9A-1300 construct of 74 nucleotides (SEQ ID NO nucleotides 487-560 of 3) and the 3' end DNA sequence (nucleotides 561-732 of SEQ ID NO: 3) of the CaMV 35S terminator of 172 nucleotides, comprising said SEQ ID NO: 3 or its complementary sequence can be identified as the existence of transgenic rice event ZUPM01.

The nucleic acid sequence may be at least 11 or more contiguous polynucleotides (third nucleic acid sequence) of any part of the transgene insertion sequence in the SEQ ID NO: 4 or its complementary sequence, or the SEQ ID NO : at least 11 or more contiguous polynucleotides (fourth nucleic acid sequence) of any part of the 3' right flank rice genomic DNA region in 4 or its complementary sequence. Said nucleic acid sequence may further be homologous or complementary to a portion of said SEQ ID NO:4 comprising the entirety of said SEQ ID NO:2. When a third nucleic acid sequence and a fourth nucleic acid sequence are used together, these nucleic acid sequences comprise a set of DNA primers in a DNA amplification method that produces an amplified product. When the amplification product generated in the DNA amplification method using the DNA primer pair is an amplification product comprising SEQ ID NO: 2, the presence of the transgenic rice event ZUPM01 or its progeny can be diagnosed.

Said SEQ ID NO: 4 or its complementary sequence is a sequence of 913 nucleotides in length near the insertion junction at the 3' end of the inserted sequence in the transgenic rice event ZUPM01, said SEQ ID NO: 4 or its complementary sequence The complementary sequence consists of a 254 nucleotide nos (nopaline synthase) transcription terminator sequence (nucleotides 1-254 of SEQ ID NO: 4), a 219 nucleotide DNA sequence of the right border of the pPHF1G9A-1300 construct (SEQ ID NO: 4 nucleotides 255-473) and 440 nucleotides of rice integration site right flank genomic DNA sequence (SEQ ID NO: 4 nucleotides 474-913), comprising the SEQ ID NO: 4 or its complementary sequence can be identified as the presence of the transgenic rice event ZUPM01.

The SEQ ID NO: 5 or its complementary sequence is a sequence of 7306 nucleotides in length characterizing the transgenic rice event ZUPM01, and its specific genome and genetic elements are shown in Table 1. The presence of the transgenic rice event ZUPM01 can be identified by including said SEQ ID NO: 5 or its complementary sequence.

Table 1 The genome and genetic elements contained in SEQ ID NO: 5

1: The unit is bp.

The nucleic acid sequence or its complementary sequence can be used in a DNA amplification method to produce an amplicon, and the detection of the amplicon diagnoses the presence of the transgenic rice event ZUPM01 or its progeny in a biological sample; the nucleic acid sequence or its complementary sequence Can be used in nucleotide detection methods to detect the presence of transgenic rice event ZUPM01 or its progeny in biological samples.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of the transgenic rice event ZUPM01 in a sample, comprising:

contacting the sample to be tested with at least two primers in a nucleic acid amplification reaction;

Perform nucleic acid amplification reactions;

detecting the presence of the amplification product;

The amplified product comprises SEQ ID NO: 1 or its complementary sequence, and/or SEQ ID NO: 2 or its complementary sequence;

The amplified product is derived from the transgenic rice event ZUPM01.

In the above technical solution, the amplification product further includes SEQ ID NO: 6 or its complementary sequence, and/or SEQ ID NO: 7 or its complementary sequence.

Specifically, the primers include a first primer and a second primer, the first primer is selected from SEQ ID NO: 1 or its complementary sequence, SEQ ID NO: 8 and SEQ ID NO: 10; the second primer is selected from From SEQ ID NO:2 or its complement, SEQ ID NO:9 and SEQ ID NO:11.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of the transgenic rice event ZUPM01 in a sample, comprising:

Contacting the sample to be detected with a probe comprising SEQ ID NO: 1 or a complementary sequence thereof, or SEQ ID NO: 2 or a complementary sequence thereof, the probe is derived from the transgenic rice event ZUPM01;

hybridize the sample to be detected and the probe under stringent hybridization conditions;

Detecting the hybridization between the sample to be detected and the probe.

The stringent conditions can be hybridized at 65° C. in 6×SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution, and then mixed with 2×SSC, 0.1% SDS and 1×SSC, Wash each membrane once with 0.1% SDS.

Further, the probe also includes SEQ ID NO: 6 or its complementary sequence, or SEQ ID NO: 7 or its complementary sequence.

Optionally, at least one of said probes is labeled with at least one fluorophore.

To achieve the above object, the present invention also provides a method for detecting the presence of DNA of the transgenic rice event ZUPM01 in a sample, comprising:

Contacting the sample to be detected with a marker nucleic acid molecule comprising SEQ ID NO: 1 or its complement, or SEQ ID NO: 2 or its complement derived from a transgenic rice event ZUPM01;

Hybridizing the sample to be detected and the marker nucleic acid molecule under stringent hybridization conditions;

Detecting the hybridization between the sample to be detected and the marker nucleic acid molecule, and then performing marker-assisted breeding analysis to determine whether herbicide tolerance and/or low phosphorus stress tolerance are genetically related to the marker nucleic acid molecule Chained.

Further, the marker nucleic acid molecule also includes SEQ ID NO: 6 or its complementary sequence, or SEQ ID NO: 7 or its complementary sequence.

To achieve the above object, the present invention also provides a DNA detection kit, comprising at least one DNA molecule, said DNA molecule comprising SEQ ID NO: 1 or its complementary sequence, or SEQ ID NO: 2 or its complementary sequence, which It can be used as one of the DNA primers or probes specific to the transgenic rice event ZUPM01 or its progeny; the DNA molecule is derived from the transgenic rice event ZUPM01.

Further, when the DNA molecule is used as a probe, it also includes SEQ ID NO: 6 or its complementary sequence, or SEQ ID NO: 7 or its complementary sequence.

To achieve the above object, the present invention also provides a method for protecting rice plants from damage caused by herbicides, comprising applying an effective dose of glyphosate herbicides to a field where at least one transgenic rice plant is planted, so that The transgenic rice plant comprises SEQ ID NO: 1, the 561-6647th nucleic acid sequence of SEQ ID NO: 5 and SEQ ID NO: 2 in its genome, or the genome of the transgenic rice plant comprises SEQ ID NO: 5 ; The transgenic rice plant has tolerance to glyphosate herbicide.

In order to achieve the above object, the present invention also provides a method for protecting rice plants from damage caused by low phosphorus elements in the soil, comprising planting at least one transgenic rice plant in soil with low phosphorus concentration, and the transgenic rice plant is planted in its The genome sequentially comprises SEQ ID NO: 1, the 561-6647 nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 2, or the genome of the transgenic rice plant comprises SEQ ID NO: 5; the transgenic rice plant It is tolerant to low phosphorus stress.

In order to achieve the above object, the present invention also provides a method of controlling weeds in the field of rice plants, comprising applying an effective dose of glyphosate herbicide to the field of planting at least one transgenic rice plant, the transgenic The rice plant sequentially includes SEQ ID NO: 1, the 561-6647th nucleic acid sequence of SEQ ID NO: 5 and SEQ ID NO: 2 in its genome, or the genome of the transgenic rice plant includes SEQ ID NO: 5; The transgenic rice plants are tolerant to glyphosate herbicide.

To achieve the above object, the present invention also provides a method for cultivating rice plants tolerant to glyphosate herbicides, comprising:

Planting at least one rice seed, the genome of the rice seed comprises a nucleic acid sequence of a specific region, and the nucleic acid sequence of the specific region comprises SEQ ID NO: 1, SEQ ID NO: 5 nucleotide sequence 561-6647 and SEQ ID NO: 2, or the nucleic acid sequence of the specific region comprises SEQ ID NO: 5;

growing said rice seeds into rice plants;

The rice plants are sprayed with an effective dose of glyphosate herbicide, and the plants with reduced plant damage compared with other plants without the nucleic acid sequence of the specific region are harvested.

To achieve the above object, the present invention also provides a method for cultivating rice plants tolerant to low phosphorus stress, characterized in that, comprising:

At least one rice seed is planted in low-phosphorus soil, the genome of the rice seed contains a nucleic acid sequence of a specific region, and the nucleic acid sequence of the specific region sequentially includes SEQ ID NO: 1, SEQ ID NO: 5 No. 561-6647 Bit nucleic acid sequence and SEQ ID NO: 2, or the nucleic acid sequence of the specific region comprises SEQ ID NO: 5;

growing said rice seeds into rice plants;

Plants having reduced plant damage compared to other plants not having the nucleic acid sequence of the specific region are harvested.

To achieve the above object, the present invention also provides a method for producing rice plants tolerant to glyphosate herbicides, comprising introducing SEQ ID NO: 5 561-6647 into the genome of the rice plants Nucleic acid sequence, and make the genome of described rice plant comprise SEQ ID NO:1, SEQ ID NO:5 the 561-6647 nucleotide sequence and SEQ ID NO:2 successively, or make the genome of described rice plant comprise SEQ ID NO : 5, selecting rice plants tolerant to glyphosate.

Specifically, the method for producing rice plants tolerant to glyphosate herbicides includes:

sexually crossing a first parent rice plant of the transgenic rice event ZUPM01 that is tolerant to glyphosate herbicides with a second parent rice plant that lacks glyphosate tolerance to generate a large number of progeny plants;

Treating the progeny plants with glyphosate herbicide;

The progeny plants are selected for tolerance to glyphosate.

In order to achieve the above object, the present invention also provides a method for producing a rice plant tolerant to low phosphorus stress, which is characterized in that it includes introducing SEQ ID NO: 5 No. 561-6647 into the genome of the rice plant nucleotide sequence, and the genome of the rice plant comprises SEQ ID NO: 1, the 561-6647th nucleic acid sequence of SEQ ID NO: 5 and SEQ ID NO: 2, or the genome of the rice plant comprises SEQ ID NO: 5, select rice plants tolerant to low phosphorus stress.

Specifically, the method for producing rice plants tolerant to low phosphorus stress includes:

Sexually crossing a first parent rice plant of the transgenic rice event ZUPM01 that is tolerant to low phosphorus stress with a second parent rice plant that lacks low phosphorus stress tolerance to produce a large number of progeny plants;

Treating the progeny plants with low phosphorus stress;

The progeny plants tolerant to low phosphorus stress are selected.

To achieve the above object, the present invention also provides a composition produced from the transgenic rice event ZUPM01, the composition is rice, straw, rice husk or rice seeds.

To achieve the above object, the present invention also provides an agricultural product or commodity produced by the transgenic rice event ZUPM01, the agricultural product or commodity is rice flour, rice oil, rice bran, rice germ, rice protein, rice starch, rice bran nutrient oil or rice bran Polysaccharides, cosmetics or fillers.

In the nucleic acid sequence and its detection method for detecting rice plants of the present invention, the following definitions and methods can better define the present invention and guide those of ordinary skill in the art to implement the present invention, unless otherwise specified, according to the ordinary skills in the art common usage by people to understand the term.

The "rice" refers to rice (Oryza sativa), including all plant varieties that can be propagated with rice, including wild Oryza species and those plants belonging to the genus Oryza that allow interspecies propagation.

The "comprising" means "including but not limited to".

The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps and whole plants in plants or plant parts Cells, said plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, and the like. Parts of transgenic plants that are understood to be within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, as well as flowers, stems, fruits, leaves and roots, the above plant parts being derived from the A transgenic plant transformed with a DNA molecule and thus consisting at least in part of a transgenic cell, or its progeny.

The term "gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences before the coding sequence (5' non-coding sequence) and regulatory sequences after the coding sequence (3' non-coding sequence). "Native gene" refers to a gene that is found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences not found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. "Exogenous gene" is a foreign gene that exists in the genome of an organism and does not exist before, and also refers to a gene that is introduced into a recipient cell through a transgenic procedure. Foreign genes may comprise native or chimeric genes inserted into a non-native organism. A "transgene" is a gene that has been introduced into the genome by a transformation procedure. The site in the plant genome where the recombinant DNA has been inserted may be referred to as the "insertion site" or "target site".

"Flanking DNA" may comprise the genome naturally present in an organism such as a plant or exogenous (heterologous) DNA introduced through a transformation process, such as a fragment associated with a transformation event. Thus, flanking DNA can include a combination of native and foreign DNA. In the present invention, "flanking region" or "flanking sequence" or "genome border region" or "genome border sequence" means at least 3, 5, 10, 11, 15, 20, 50, 100, 200, 300, 400 , 1000, 1500, 2000, 2500, or 5000 base pairs or longer of sequence that is located immediately upstream or downstream of and adjacent to the original exogenously inserted DNA molecule. When the flanking region is located upstream, it may also be referred to as "left border flank" or "5' flank" or "5' genomic flanking region" or "genomic 5' flanking sequence" and the like. When the flanking region is located downstream, it may also be referred to as "right border flanking" or "3' flanking" or "3' genomic flanking region" or "genomic 3' flanking sequence" and the like.

Transformation procedures that result in random integration of exogenous DNA will result in transformants containing different flanking regions that are specific to each transformant. When recombinant DNA is introduced into plants by conventional crossing, its flanking regions are usually not altered. Transformants will also contain unique junctions between the heterologous insert DNA and the segment of genomic DNA or between two segments of genomic DNA or between two segments of heterologous DNA. A "junction" is the point at which two specific DNA segments join. For example, junctions exist where insert DNA joins flanking DNA. Junctions also exist in transformed organisms where two segments of DNA join together in a manner modified from that found in natural organisms. "Junction DNA" refers to DNA comprising a junction.

The present invention provides a transgenic rice event called ZUPM01 and progeny thereof, said transgenic rice event ZUPM01 is rice plant ZUPM01, which includes plants and seeds of transgenic rice event ZUPM01 and plant cells or regenerable parts thereof, said transgenic Plant parts of rice event ZUPM01, including but not limited to cells, pollen, ovules, flowers, buds, roots, stems, ears, inflorescences, leaves and products from rice plant ZUPM01, such as rice, rice straw, rice hulls or rice seeds and leaves Biomass in a rice crop field.

The transgenic rice event ZUPM01 of the present invention comprises a DNA construct, and when expressed in plant cells, said transgenic rice event ZUPM01 acquires tolerance to glyphosate herbicide and/or low phosphorus stress. The DNA construct comprises an expression cassette comprising a suitable promoter for expression in plants and a suitable polyadenylation signal sequence, said promoter being operably linked to a protein encoding 5-enol-acetone A gene of shikimate-3-phosphate synthase (G9A), the nucleic acid sequence of the G9A protein has tolerance to glyphosate herbicide. The DNA construct comprises a further expression cassette comprising a suitable promoter for expression in plants and a suitable polyadenylation signal sequence, the promoter being operably linked to a protein encoding the rice phosphate transporter Body Facilitated Transport Factor (OsPHF1) gene, the nucleic acid sequence of the OsPHF1 protein has tolerance to low phosphorus stress. Further, the promoter may be a suitable promoter isolated from a plant, including a constitutive, inducible and/or tissue-specific promoter, and the suitable promoter includes, but is not limited to, cauliflower mosaic virus (CaMV) 35S Promoter, Scrophulariaceae mosaic virus (FMV) 35S promoter, Ubiquitin promoter, Actin promoter, Agrobacterium tumefaciens nopaline synthase (NOS) promoter, Octopine synthase (OCS) promoter, Cestrum yellow leaf curl virus promoter, potato tuber storage protein (Patatin) promoter, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter, glutathione sulfur transferase (GST) promoter, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes (Agrobacterium rhizogenes) RolD promoter and Arabidopsis thaliana ) Suc2 promoter. The polyadenylation signal sequence may be a suitable polyadenylation signal sequence that functions in plants, and the suitable polyadenylation signal sequence includes, but is not limited to, derived from Agrobacterium tumefaciens The polyadenylation signal sequence of the nopaline synthase (NOS) gene, the 35S terminator from the cauliflower mosaic virus (CaMV), the polyadenylation signal sequence from the protease inhibitor II (PIN II) gene and Polyadenylation signal sequence derived from the α-tubulin gene.

In addition, the expression cassette may also include other genetic elements including, but not limited to, enhancers and signal/transit peptides. The enhancer can enhance the expression level of the gene, and the enhancer includes, but is not limited to, tobacco etch virus (TEV) translation activator, CaMV35S enhancer and FMV35S enhancer. The signal peptide/transit peptide can guide the G9A protein to be transported to a specific organelle or compartment outside the cell or within the cell, for example, using the sequence encoding the chloroplast transit peptide to target the chloroplast, or using the 'KDEL' retention sequence to target the endoplasmic reticulum.

The "glyphosate" refers to N-phosphonomethylglycine and its salts, and the treatment with "glyphosate herbicide" refers to the use of any herbicide formulation containing glyphosate. The selection of the application rate of a glyphosate formulation to achieve a biologically effective dose is within the skill of the ordinary agronomist. Treatment of a field containing plant material derived from transgenic rice event ZUPM01 with any of the glyphosate-containing herbicide formulations will control weed growth in said field without affecting plant material derived from transgenic rice event ZUPM01 growth or yield.

The DNA construct is introduced into the plant using a transformation method including, but not limited to, Agrobacterium-mediated transformation, biolistic transformation, and pollen tube passage transformation.

The Agrobacterium-mediated transformation method is a common method for plant transformation. The foreign DNA to be introduced into the plant is cloned between the left and right border consensus sequences of the vector, ie the T-DNA region. The vector is transformed into Agrobacterium cells, which are then used to infect plant tissues, and the T-DNA region of the vector containing foreign DNA is inserted into the plant genome.

The gene bombardment transformation method is bombarding plant cells with a vector containing foreign DNA (particle-mediated biolistic transformation).

The pollen tube channel transformation method utilizes the natural pollen tube channel (also known as pollen tube guiding tissue) formed after plant pollination to carry exogenous DNA into the embryo sac through the nucellus channel.

Following transformation, transgenic plants must be regenerated from the transformed plant tissue, and appropriate markers used to select for progeny bearing the foreign DNA.

A DNA construct is an interconnected assembly of DNA molecules that provides one or more expression cassettes. The DNA construct is preferably a plasmid capable of self-replicating in bacterial cells and containing various restriction endonuclease sites for introduction to provide a functional genetic element, i.e. a promoter , introns, leader sequences, coding sequences, 3' terminator regions and other sequences of DNA molecules. The expression cassette contained in the DNA construct includes the genetic elements necessary to provide transcription of the messenger RNA and can be designed for expression in prokaryotic or eukaryotic cells. The expression cassettes of the invention are designed for expression most preferably in plant cells.

A transgenic "event" is obtained by transforming a plant cell with a heterologous DNA construct, i.e., comprising at least one nucleic acid expression cassette containing a gene of interest, inserted transgenically into the plant genome to produce a plant population, which is regenerated , and select specific plants with features inserted into specific genomic loci. The term "event" refers to the original transformant comprising heterologous DNA and the progeny of that transformant. The term "event" also refers to the progeny of a sexual cross between a transformant and an individual of another breed containing heterologous DNA, even after repeated backcrosses with the backcross parent, the insert DNA and flanking Genomic DNA is also present at the same chromosomal location in hybrid offspring. The term "event" also refers to a DNA sequence from an original transformant comprising the insert DNA and flanking genomic sequences in close proximity to the insert DNA, which DNA sequence is expected to be transferred to progeny derived from cells containing the insert DNA The parental line (such as the original transformant and its progeny produced by selfing) is sexually crossed with a parental line that does not contain the inserted DNA, and the progeny receive the inserted DNA containing the gene of interest.

"Recombinant" in the present invention refers to a form of DNA and/or protein and/or organism that is not normally found in nature and thus produced by human intervention. Such human intervention can produce recombinant DNA molecules and/or recombinant plants. Said "recombinant DNA molecule" is obtained by the artificial combination of two otherwise separate sequence segments, such as by chemical synthesis or by manipulation of separate nucleic acid segments by genetic engineering techniques. The techniques for performing nucleic acid manipulations are well known.

The term "transgenic" includes any cell, cell line, callus, tissue, plant part or plant, the genotype of which is altered by the presence of heterologous nucleic acid, said "transgenic" including transgenics originally so altered as well as those derived from The offspring individuals produced by the original transgenic body through sexual crossing or asexual reproduction. In the present invention, the term "transgenic" does not include alterations of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods or naturally occurring events such as random heterozygous fertilization, non-recombinant viral infection, non-recombinant Bacterial transformation, non-recombinant transposition, or spontaneous mutation.

"Heterologous" in the context of the present invention means that a first molecule is not normally found in combination with a second molecule in nature. For example, a molecule can be derived from a first species and inserted into the genome of a second species. This molecule is thus heterologous to the host and is artificially introduced into the genome of the host cell.

The transgenic rice event ZUPM01 tolerant to glyphosate herbicides was grown by first sexually crossing a first parent rice plant with a second parent rice plant to generate diverse first generation progeny plants, The first parent rice plants consist of rice plants bred from the transgenic rice event ZUPM01 and its progeny, which are tolerant to glyphosate herbicides and/or low phosphorus stress by using the glyphosate herbicide and/or low phosphorus stress of the present invention The second parent rice plant lacks tolerance to glyphosate herbicide and/or low phosphorus stress; and then selects to have tolerance to glyphosate herbicide and/or low phosphorus stress The tolerant progeny plants can be used to breed rice plants tolerant to glyphosate herbicides and/or low phosphorus stress. These steps may further comprise backcrossing progeny plants tolerant to glyphosate and/or low phosphorus stress to second parent rice plants or third parent rice plants, followed by herbicide application with glyphosate or low phosphorus Stress or by trait-related molecular markers such as DNA molecules comprising the junction sites identified at the 5' end and 3' end of the inserted sequence in the transgenic rice event ZUPM01) to select progeny, thereby producing glyphosate-resistant Rice plants tolerant to herbicide and/or low phosphorus stress.

It should also be understood that two different transgenic plants can also be crossed to produce progeny that contain two independent, segregated additions of the exogenous gene. Selfing of suitable progeny can result in progeny plants that are homozygous for both added exogenous genes. Backcrossing to parental plants and outcrossing to non-transgenic plants are also contemplated as previously described, as is vegetative propagation.

The term "probe" is an isolated nucleic acid molecule to which is bound a conventional detectable label or reporter molecule, eg, a radioisotope, ligand, chemiluminescent agent or enzyme. This probe is complementary to one strand of the target nucleic acid, and in the present invention, the probe is complementary to one strand of DNA from the genome of the transgenic rice event ZUPM01, whether the genomic DNA is from the transgenic rice event ZUPM01 or the seed or is derived from the transgene Plants or seeds or extracts of rice event ZUPM01. The probes of the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that specifically bind to a target DNA sequence and can be used to detect the presence of the target DNA sequence.

The term "primer" is an isolated nucleic acid molecule that, by nucleic acid hybridization, anneals to a complementary target DNA strand, forms a hybrid between the primer and target DNA strand, and then reacts with a polymerase (eg, DNA polymerase) Bottom, stretches along the target DNA strand. The primer pairs of the present invention relate to their use in the amplification of target nucleic acid sequences, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

Probes and primers are generally 11 polynucleotides or more in length, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or more, most preferably 30 polynucleotides in length sour or more. Such probes and primers hybridize specifically to the target sequence under highly stringent hybridization conditions. Although probes that are different from the target DNA sequence and maintain the ability to hybridize to the target DNA sequence can be designed by conventional methods, preferably, the probes and primers in the present invention have complete DNA sequences with the continuous nucleic acid of the target sequence identity.

The primers and probes based on the flanking genomic DNA and the insert sequence of the present invention can be determined by conventional methods, for example, by isolating the corresponding DNA molecule from the plant material derived from the transgenic rice event ZUPM01, and determining the nucleic acid sequence of the DNA molecule. The DNA molecule contains a transgene insertion sequence and rice genome flanking regions, and the fragments of the DNA molecule can be used as primers or probes.

The nucleic acid probes and primers of the invention hybridize to target DNA sequences under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA derived from transgenic rice event ZUPM01 in a sample. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be capable of specifically hybridizing to each other if the two nucleic acid molecules are capable of forming an antiparallel double-stranded nucleic acid structure. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if two nucleic acid molecules exhibit perfect complementarity. As used herein, two nucleic acid molecules are said to exhibit "perfect complementarity" when every nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to be "complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "high stringency" conditions. Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the particular solvent and salt concentration employed.

As used herein, a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing to a matching complementary strand of another nucleic acid molecule under highly stringent conditions. Suitable stringent conditions to promote DNA hybridization, for example, treatment with 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by washing with 2.0× SSC at 50° C., are known to those skilled in the art. is well known. For example, the salt concentration in the washing step can be selected from about 2.0×SSC, 50°C for low stringency conditions to about 0.2×SSC, 50°C for high stringency conditions. In addition, the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions. Both the temperature condition and the salt concentration can be changed, or one can be kept constant while the other variable is changed. Preferably, a nucleic acid molecule of the present invention can be combined with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4. One or more nucleic acid molecules in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 or their complementary sequences, or any fragment of the above sequences specifically hybridize. More preferably, a nucleic acid molecule of the present invention is combined with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 under highly stringent conditions Specific hybridization occurs with one or more nucleic acid molecules in SEQ ID NO: 7 or its complementary sequence, or any fragment of the above sequence. In the present invention, the preferred marker nucleic acid molecule has SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 7 or its complementary sequence, or any fragment of the above sequence.

Another preferred marker nucleic acid molecule of the present invention has 80% to 100% or 90% to 100% sequence identity. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 7 can be used as markers in plant breeding methods to identify progeny of genetic crosses. The hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including but not limited to fluorescent labeling, radioactive labeling, antibody-based labeling and chemiluminescent labeling.

With respect to the amplification of a target nucleic acid sequence using specific amplification primers (for example, by PCR), "stringent conditions" refer to conditions that allow only the primers to hybridize to the target nucleic acid sequence in a DNA thermal amplification reaction, with the same The primer corresponding to the wild-type sequence (or its complementary sequence) of the target nucleic acid sequence is capable of binding to the target nucleic acid sequence, and preferably produces a unique amplification product, the amplification product being an amplicon.

The term "specifically binds (to a target sequence)" means that under stringent hybridization conditions a probe or primer hybridizes only to a target sequence in a sample containing the target sequence.

As used herein, "amplified DNA" or "amplicon" refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. For example, in order to determine whether rice plants are produced by sexual crossing containing the transgenic rice event ZUPM01 of the present invention, or whether rice samples collected from the field contain the transgenic rice event ZUPM01, or whether rice extracts, such as meal, meal or oil contain DNA extracted from rice plant tissue samples or extracts of transgenic rice event ZUPM01 can be subjected to nucleic acid amplification methods using primer pairs to generate amplicons that are diagnostic for the presence of DNA from transgenic rice event ZUPM01. The pair of primers includes a first primer derived from a flanking sequence adjacent to the insertion site of the inserted foreign DNA in the plant genome, and a second primer derived from the inserted foreign DNA. The amplicon has a length and sequence that is also diagnostic for the transgenic rice event ZUPM01.

The length of the amplicon may range from the combined length of the primer pair plus one nucleotide base pair, preferably plus about fifty nucleotide base pairs, more preferably plus about two hundred and fifty nucleotides base pairs, most preferably plus about four hundred and fifty nucleotide base pairs or more.

Alternatively, primer pairs can be derived from flanking genomic sequences flanking the insert DNA to generate amplicons that include the entire insert nucleotide sequence. One of the primer pairs derived from the plant genomic sequence can be located at a distance from the insert DNA sequence, which distance can range from one nucleotide base pair to about twenty thousand nucleotide base pairs. The use of the term "amplicon" specifically excludes primer-dimers formed in DNA thermal amplification reactions.

Nucleic acid amplification reaction can be achieved by any nucleic acid amplification reaction method known in the art, including polymerase chain reaction (PCR). Various nucleic acid amplification methods are well known to those skilled in the art. PCR amplification methods have been developed to amplify 22kb of genomic DNA and 42kb of phage DNA. These methods, as well as other DNA amplification methods known in the art, can be used in the present invention. The inserted exogenous DNA sequence and the flanking DNA sequence from the transgenic rice event ZUPM01 can be amplified by using the primer sequences provided to the genome of the transgenic rice event ZUPM01, and after the amplification, standard PCR amplicons or cloned DNA DNA sequencing.

DNA detection kits based on DNA amplification methods contain DNA primer molecules that, under appropriate reaction conditions, specifically hybridize to target DNA and amplify diagnostic amplicons. Kits may provide agarose gel-based detection methods or a number of methods known in the art to detect diagnostic amplicons. Kit containing DNA primers homologous or complementary to any part of the rice genome region of SEQ ID NO: 3 or SEQ ID NO: 4, and to any part of the transgene insertion region of SEQ ID NO: 5 provided by the present invention. A primer pair specifically identified as useful in DNA amplification methods is SEQ ID NO: 8 and SEQ ID NO: 9, which amplifies diagnostic amplification of a portion of the 5' transgene/genomic region homologous to the transgenic rice event ZUPM01 sub, wherein the amplicon comprises SEQ ID NO: 1. Primer pairs identified as useful in DNA amplification methods also include SEQ ID NO: 10 and SEQ ID NO: 11, which amplify a diagnostic amplicon homologous to a portion of the 3' transgene/genomic region of transgenic rice event ZUPM01 , wherein the amplicon comprises SEQ ID NO:2. Other DNA molecules used as DNA primers may be selected from SEQ ID NO:5.

Amplicons generated by these methods can be detected by a variety of techniques. One such method is GeneticBit Analysis, which designs a DNA oligonucleotide strand spanning the insert DNA sequence and the adjacent flanking genomic DNA sequence. The oligonucleotide strands are immobilized in the microwells of a microplate, and after PCR amplification of the region of interest (using one primer each within the insert sequence and adjacent flanking genomic sequences), the single-stranded PCR product Can hybridize to immobilized oligonucleotide strands and serve as templates for single-base extension reactions using a DNA polymerase and ddNTPs specifically labeled for the next expected base. Results can be obtained by fluorescent or ELISA-like methods. The signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization and single base extension reactions were successful.

Another method is Pyrosequencing (pyrosequencing) technology. This method designs an oligonucleotide strand that spans the insert DNA sequence and the adjacent genomic DNA binding site. This oligonucleotide strand is hybridized to a single-stranded PCR product of the region of interest (using one primer each within the insert and adjacent flanking genomic sequences), followed by DNA polymerase, ATP, sulfurylase, luciferin The enzyme, apyrase, adenosine-5'-phosphosulfate and luciferin are incubated together. Add dNTPs respectively, and measure the light signal generated. The light signal represents the presence of the insertion/flanking sequence, which indicates that the amplification, hybridization, and single-base or multi-base extension reactions were successful.

Fluorescence polarization is also a method that can be used to detect amplicons of the present invention (Chen X, Levine L, and Kwok P Y. Fluorescence polarization in homogeneous nucleic acid analysis [J]. Genome Res, 1999, 9 (5): 492-8.). Using this method requires the design of an oligonucleotide strand that spans the insert DNA sequence and the adjacent genomic DNA binding site. The oligonucleotide strand is hybridized to a single-stranded PCR product of the region of interest (using one primer each within the insert and adjacent flanking genomic sequence) with DNA polymerase and a fluorescently labeled ddNTP Incubation. Single base extensions result in the insertion of ddNTPs. This insertion can be measured as a change in polarization using a fluorometer. A change in polarization represents the presence of insertion/flanking sequences, which indicates that the amplification, hybridization and single base extension reactions were successful.

Taqman is described as a method for the detection and quantification of the presence of DNA sequences, which is described in detail in the instructions for use provided by the manufacturer. As a brief example, design a FRET oligonucleotide probe spanning the insertion DNA sequence and the adjacent genomic flanking junction site as follows. The FRET probe and PCR primers (one each within the insert and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage of the fluorescent and quencher moieties on the FRET probe and release of the fluorescent moiety. The generation of a fluorescent signal represents the presence of the insertion/flanking sequence, which indicates that the amplification and hybridization were successful.

Based on hybridization principles, suitable techniques for detecting plant material derived from transgenic rice event ZUPM01 may also include Southern blot hybridization, Northern blot hybridization and in situ hybridization. In particular, such suitable techniques include incubating the probe and sample, washing to remove unbound probe and detecting whether the probe has hybridized. The detection method depends on the type of label attached to the probe, for example, radiolabeled probes can be detected by X-ray film exposure and visualization, or enzyme-labeled probes can be detected by substrate conversion to achieve a color change.

Molecular markers can also be used to detect sequences (Tyagi S and Kramer F R. Molecular beacons: probes that fluoresce upon hybridization [J]. Nat Biotechnol, 1996, 14(3): 303-8.). Design a FRET oligonucleotide probe that spans the insertion DNA sequence and the adjacent genomic flanking junction site. The unique structure of this FRET probe results in it containing a secondary structure that is capable of maintaining a fluorescent moiety and a quencher moiety in close proximity. The FRET probe and PCR primers (one each within the insert and adjacent flanking genomic sequences) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, the hybridization of the FRET probe to the target sequence leads to the loss of the secondary structure of the probe, thereby spatially separating the fluorescent part and the quencher part, resulting in a fluorescent signal. The generation of a fluorescent signal represents the presence of the insertion/flanking sequence, which indicates that the amplification and hybridization were successful.

Other described methods such as microfluidics provide methods and devices for isolating and amplifying DNA samples. Optical dyes are used to detect and measure specific DNA molecules. Nanotube devices comprising electronic sensors for the detection of DNA molecules or nanobeads which bind specific DNA molecules and thus can be detected are useful for the detection of the DNA molecules of the present invention.

DNA detection kits can be developed using the compositions described herein and methods described or known in the field of DNA detection. The kit is beneficial for identifying whether the DNA of the transgenic rice event ZUPM01 exists in a sample, and can also be used for cultivating rice plants containing the DNA of the transgenic rice event ZUPM01. The kit may contain DNA primers or probes homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4 or 5, or other DNA primers or probes homologous to or complementary to at least a portion of SEQ ID NO: 1, 2, 3, 4 or 5 The DNA contained in the transgenic genetic element is complementary to the DNA, which DNA sequences can be used in DNA amplification reactions, or as probes in DNA hybridization methods. The DNA structure of the transgene insertion sequence and the rice genome binding site contained in the rice genome and illustrated in Figure 1 and Table 1 contains: the rice ZUPM01 left flank genomic region located at the 5' end of the transgene insertion sequence, from the left of Agrobacterium A part of the insert sequence in the side border region (LB), the first expression cassette is operably linked to the signal peptide sequence (AHAS) of the maize acetolactate synthase gene by the maize ubiquitin gene promoter (ubiquitin promoter), which can Operably linked to the glyphosate-tolerant 5-enol-pyruvylshikimate-3-phosphate synthase gene (G9A) in R. radiodurans and operably linked to the 35S terminator of cauliflower mosaic virus The second expression cassette consists of the cauliflower mosaic virus 35S promoter and the maize ubiquitin gene promoter (CaMV 355 promoter), operably linked to the rice The phosphate transporter facilitates the transport factor sequence (OsPHF1) and is operably linked to the nopaline synthase gene terminator (nos terminator), a part of the insertion sequence from the right border region (RB) of Agrobacterium, and the right flank genome region of rice plant ZUPM01 (SEQ ID NO: 5) located at the 3' end of the transgene insertion sequence. In the DNA amplification method, the DNA molecules used as primers can be derived from any part of the transgene insertion sequence in the transgenic rice event ZUPM01, and can also be derived from any part of the DNA region of the flanking rice genome in the transgenic rice event ZUPM01.

The transgenic rice event ZUPM01 can be combined with other transgenic rice varieties, such as herbicide (such as glufosinate, dicamba, etc.) tolerant rice, or transgenic rice varieties carrying insect resistance genes. Various combinations of all these different transgenic events, bred together with the transgenic rice event ZUPM01 of the present invention, can provide improved hybrid transgenic rice varieties resistant to insects and various herbicides and tolerant to low phosphorus stress. Compared with non-transgenic varieties and single-trait transgenic varieties, these varieties can show more excellent characteristics such as yield improvement.

The invention provides a nucleic acid sequence for detecting rice plants and a detection method thereof. The transgenic rice event ZUPM01 has the function of tolerance to agricultural herbicides containing glyphosate and/or low phosphorus stress. The rice plants with this trait express glyphosate-resistant R. radiodurans 5-enol-pyruvylshikimate-3-phosphate synthase (G9A) protein and rice phosphate transporter facilitator protein (OsPHF1), which Confers tolerance to glyphosate and low phosphorus stress in plants. At the same time, the detection method of the present invention comprises a sequence of SEQ ID NO: 1 or its complementary sequence, a sequence comprising SEQ ID NO: 2 or its complementary sequence, SEQ ID NO: 6 or its complementary sequence, or SEQ ID NO: 7 or its complementary sequence The complementary sequence can be used as a DNA primer or probe to generate an amplified product that is diagnosed as the transgenic rice event ZUPM01 or its progeny, and can quickly, accurately and stably identify the presence of plant materials derived from the transgenic rice event ZUPM01.

Sequence brief:

SEQ ID NO: 11 nucleotide sequences of rice genomic DNA on the left flank of the 5' transgene insertion site in 1 transgenic rice event ZUPM01 and the left border of the transgene fragment;

SEQ ID NO: 11 nucleotide sequences of the right border of the transgene fragment at the 3' transgene insertion site in 2 transgenic rice event ZUPM01 and the right flank rice genomic DNA;

SEQ ID NO: 3 A sequence of 732 nucleotides in length near the insertion junction at the 5' end of the insertion sequence in the transgenic rice event ZUPM01;

SEQ ID NO: 4 A sequence of 913 nucleotides in length near the insertion junction at the 3' end of the insertion sequence in the transgenic rice event ZUPM01;

SEQ ID NO: 5 5' left flank rice genome sequence, whole T-DNA sequence and 3' right flank rice genome sequence;

SEQ ID NO: 6 is located in the sequence within SEQ ID NO: 3, spanning the 5' left flank rice genome sequence, pPHF1G9A-1300 construct left border DNA sequence and CaMV 35S terminator sequence;

SEQ ID NO: 7 is located in the sequence within SEQ ID NO: 4, spanning the nos transcription termination sequence, the right border DNA sequence of the pPHF1G9A-1300 construct and the 3' right flank rice genome sequence;

SEQ ID NO: 8 amplifies the first primer of SEQ ID NO: 6;

SEQ ID NO: 9 amplifies the second primer of SEQ ID NO: 6;

SEQ ID NO: 10 amplifies the first primer of SEQ ID NO: 7;

SEQ ID NO: 11 amplifies the second primer of SEQ ID NO: 7;

SEQ ID NO: 12 The first primer for PCR detection of G9A;

SEQ ID NO: 13 The second primer for PCR detection of G9A;

SEQ ID NO: 14 The first primer for PCR detection of OsPHF1;

SEQ ID NO: 15 The second primer for PCR detection of OsPHF1;

SEQ ID NO: 16 Primer for obtaining the right border flanking sequence;

SEQ ID NO: 17 A primer for obtaining the right border flanking sequence;

SEQ ID NO: 18 A primer for obtaining the right border flanking sequence;

SEQ ID NO: 19 A primer for obtaining the right border flanking sequence;

SEQ ID NO: 20 A primer for obtaining the right border flanking sequence;

SEQ ID NO: 21 A primer for obtaining the right border flanking sequence;

SEQ ID NO: 22 A primer for obtaining the right border flanking sequence;

SEQ ID NO: 23 A primer for obtaining the right border flanking sequence;

SEQ ID NO: 24 A primer for obtaining the right border flanking sequence;

SEQ ID NO: 25 Primer for obtaining the right border flanking sequence;

SEQ ID NO: 26 Primer for obtaining the right border flanking sequence;

SEQ ID NO: 27 A primer for obtaining the right border flanking sequence;

SEQ ID NO: 28 A primer for obtaining the right border flanking sequence;

SEQ ID NO: 29 A primer for obtaining the right border flanking sequence;

The probe in SEQ ID NO:30 Southern hybridization detection;

The probe in SEQ ID NO: 31 Southern hybridization detection;

The technical solutions of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

Description of drawings

Fig. 1 Schematic diagram of the structure of the binding site between the transgene insertion sequence and the rice genome.

Fig. 2 The physical map of the recombinant expression vector pPHF1G9A-1300. The English and abbreviation meanings of each component are listed as follows:

CaMV 35S promoter The 35S promoter of cauliflower mosaic virus (CaMV).

OsPHF1 encodes a phosphate transporter facilitator transport factor that promotes phosphorus uptake and transport in rice.

nos terminator The terminator of the nopaline synthase gene.

T-Border(right) T-DNA right border sequence of Agrobacterium C58, required for T-DNA transfer.

pVS1 sta Plasmid stabilization site for the pVS1 plasmid.

pVS1 rep Origin of replication of the pVS1 plasmid.

pBR322 bom The bom site of the pBR322 plasmid is the action site of the migration protein mop.

pBR322 ori The origin of replication of the pBR322 plasmid.

kanamycin(R) encodes an aminoglycoside phosphotransferase protein that confers kanamycin resistance to bacteria.

T-Border(left) T-DNA left border sequence of Agrobacterium C58, required for T-DNA transfer.

CaMV 35S terminator The 35S terminator of cauliflower mosaic virus (CaMV).

G9A is derived from radiodurans and encodes an EPSPS protein that confers glyphosate resistance.

AHAS Signal peptide sequence of maize acetolactate synthase gene.

ubiquitin promoter The promoter of maize ubiquitin gene.

Fig. 3 Southern blot hybridization diagram of the inserted copy number of ZUPM01 target gene OsPHF1. A: OsPHF1 probe hybridization map; B: Schematic diagram of restriction sites and probe positions in the T-DNA region. The numbers indicate the band size after digestion, in kb, and the positions of the probes used are marked in the lower part with lines. Lanes 1-25 represent different DNA samples, respectively. 1, 7, 13, 19, 20: DNAMarker, the band size is marked on the far left, in kb; 2, 8, 14, 21: blank; 3, 9, 22: plasmid DNA; 4, 10, 15, 16 , 23: non-transgenic 9311 genomic DNA; 5, 11, 17, 24: ZUPM01 T ₅ genomic DNA; 6, 12, 18, 25: ZUPM01 T ₆ genomic DNA; 3-6: EcoRI digestion; 9-12: PvuII Enzyme digestion; 15-18: NheI digestion; 22-25: HindIII digestion.

Fig. 4 Southern blot hybridization diagram of the inserted copy number of ZUPM01 target gene G9A. A: G9A probe hybridization map; B: Schematic diagram of restriction sites and probe positions in the T-DNA region. The numbers indicate the size of the bands after digestion, in kb, and the positions of the probes used are marked in the lower part with lines. Lanes 1-18 represent different DNA samples, respectively. 1, 7, 13: DNA Marker, the band size is marked on the far left, in kb; 2, 8, 14: blank; 3, 9, 15: plasmid DNA; 4, 10, 16: non-transgenic 9311 genomic DNA; 5, 11, 17: ZUPM01 T ₅ genomic DNA; 6, 12, 18: ZUPM01 T ₆ genomic DNA; 3-6: NheI digestion; 9-12: HindIII digestion; 15-18: SacI digestion.

Figure 5 Detection results of transformation event ZUPM01. A: Left boundary detection, the expected size of the target band is 682bp; B: Right boundary detection, the expected size of the target band is 835bp; M: Molecular weight standards, from top to bottom are 2kb, 1kb, 750bp, 500bp, 250bp, 100bp; N : negative control wild-type 9311; P: negative control pPHF1G9A-1300 plasmid; W: water; T ₅ , T ₆ : respectively represent transformation events of T ₅ and T ₆ generations;

Detailed ways

The transformation event ZUPM01 involved in this application refers to a rice plant in which a foreign gene insert (T-DNA insert) is inserted between specific genome sequences through genetic transformation using rice inbred line 9311 as a recipient. In a specific embodiment, the expression vector used for the transgene has the physical map shown in FIG. 1 , and the obtained T-DNA insert has the sequence shown in nucleotides 487-6866 of SEQ ID NO:5. The transformation event ZUPM01 may refer to this transgenic process, or it may refer to the T-DNA insertion in the genome obtained by this process, or the combination of T-DNA insert and flanking sequence, or it may refer to the rice plants. In a specific example, this event is also applicable to plants obtained by transformation of other recipient species with the same expression vector, thereby inserting the T-DNA insert at the same genomic location. The transformation event ZUPM01 may also refer to offspring plants obtained by asexual reproduction, sexual reproduction, reduction or doubling reproduction or a combination of the above plants.

Example 1 Construction of transformation vector

The skeleton vector used in the present invention is the binary Ti vector p1300-UBQAHAS-1174s transformed from the pCAMBIA1300 vector, and the vector already contains the G9A gene expression cassette. The OsPHF1 gene expression cassette was constructed by In-Fusion technology. According to the cDNA sequence of rice phosphate transporter transport facilitator factor 1 (OsPHF1) gene provided by TIGR website (http://www.tigr.org/), primers were designed, and the root cDNA of japonica rice Nipponbare (Nipponbare) was used as a template to amplify After the coding region of the OsPHF1 gene was obtained, it was cloned into the pMD-19 vector (35S:PHF1:NOS/CPB), using the vector as a template, using primers containing HindIII restriction sites at both ends to amplify to obtain HindIII enzymes at both ends Cut site 35S:PHF1:NOS expression cassette product. Then the p1300-UBQAHAS-1174s vector and 35S:PHF1:NOS product were digested with HindIII, and the product was placed in the In-Fusion reaction solution and incubated at 42°C for 30 minutes, then transformed into Escherichia coli, and the ones verified by PCR and enzyme digestion were selected. Monoclonal to obtain the expression vector named pPHF1G9A-1300.

The size of the obtained vector is 12.684kb, and the physical map of the vector is shown in FIG. 1 .

Example 2 Genetic Transformation of Rice

The invention uses the Agrobacterium-mediated method to transfer the target gene expression vector into rice, and the Agrobacterium strain used is EHA105. The rice seeds were sterilized to induce callus, and then co-cultured with Agrobacterium to infect the callus, and the transformed callus was screened by selective culture, and then the plants were regenerated on the selective medium. The specific process is as follows:

1) Induction of rice callus: Take mature rice seeds, manually or mechanically dehull, select plump, smooth seeds without plaque, put them into a 100ml sterile beaker, pour 70% alcohol (15ml) into a sterilized 2min; pour off the alcohol , add 100ml of 30% sodium hypochlorite (NaClO) solution, soak for 30 minutes; pour off the sodium hypochlorite solution, wash the seeds with sterile distilled water 4-5 times, and soak for 30 minutes for the last time. Put the seeds on sterile filter paper to blot dry, put them into the mature embryo induction medium, 20-30 seeds per dish; seal the petri dish with a sealing film (Micropore ^TM Surgical Tape) after the operation, and incubate in a light incubator at 28°C . It takes 7 to 10 days to induce callus under dark culture conditions; open the petri dish on the ultra-clean workbench, pick up naturally divided embryogenic callus (light yellow, dense and spherical) with tweezers, and put it into the successor. Subculture in the subculture medium for 1 week at 28°C in the dark.

2) Agrobacterium culture: Pick the single clone of Agrobacterium transformed with the target expression vector into 15ml of YEP culture medium (containing corresponding antibiotics), culture at 28°C and 250rpm for 12-16h with shaking until the OD ₆₀₀ of the bacterial solution is 0.8-1.0.

3) Co-cultivation and selection of resistant callus: centrifuge the cultured bacterial solution at 4000 rpm for 10 min at room temperature, and remove the supernatant. Pick out the rice callus that has grown to a certain size, put it into the Agrobacterium suspension, and culture it on a horizontal shaker at 80 rpm for 30 minutes; take out the callus, put it on sterile filter paper and drain it for 30-40 minutes; put the callus Tissues were plated on co-culture medium with a sterile filter paper and incubated in the dark at 25°C for 3 days.

4) Selective culture: the callus is taken out, washed with sterile water for 5-6 times, and shaken constantly. Then wash twice with sterile water containing 300 mg/L carbenicillin sodium (Carb), shake each time on a horizontal shaker for 30 min, and finally place it on sterile filter paper to drain for 2 hours. Transfer the dried callus to the selection medium containing 300mg/L carbenicillin sodium (Carb) and the corresponding selection pressure, culture in dark at 28°C for 14 days, and carry out two rounds of selection until the granular resistant callus Tissue grows.

5) Induction of differentiation and rooting of resistant calli: Pick 3-5 resistant calli with bright yellow color from different calli, move them into plastic jars with differentiation medium, cover them with a sealing film Seal it well, put it in a constant temperature (25°C) culture room (16h/8h), and wait for it to differentiate into seedlings (about 40 days). When the seedlings grow to about 3 cm, cut off the old roots and callus from the base of the seedlings with scissors, and put them into the rooting medium to strengthen the seedlings (about 1 week).

6) Seedling hardening and transplanting: Pick out the test tubes with well-differentiated roots, stems and leaves, open the sealing film, add appropriate amount of distilled water or sterile water (to prevent the growth of bacteria in the medium), harden the seedlings for 2 to 3 days, and then wash off the agar , transplant the seedlings to the soil pot in the greenhouse, and detect the transgenic plants positive.

Example 3 Screening of Transformants

(1) Molecular detection of the target gene was carried out on the 110 T0 transformed seedlings obtained through transformation. Design two PCR primer pairs according to the two gene sequences, the primer sequences of the first primer pair are SEQ ID NO: 12 and SEQ ID NO: 13, and the primer sequences of the second primer pair are SEQ ID NO: 14 and SEQ ID NO: 15. Genomic DNA was extracted from transformed seedling leaves, and amplified according to the following PCR parameters:

reaction system:

Reaction procedure:

The sizes of the amplified fragments of the two target genes of the positive transformants were consistent with those of the positive plasmid control PCR, which were 600bp and 296bp respectively, and the T1 seeds _of 77 positive individual plants were harvested.

(2) The plants of the T ₁ -T ₃ generation were screened by herbicides, and the segregation ratio was screened to obtain 4 No. OsPHF1-9311-1, OsPHF1-9311-2, OsPHF1-9311-3, OsPHF1-9311-4 strain.

(3) Initial identification of agronomic characters of T ₄ -T ₅ generation OsPHF1-9311-1, OsPHF1-9311-2, OsPHF1-9311-3, OsPHF1-9311-4 transformants in the field, to determine OsPHF1-9311-1 ( That is, ZUPM01) has outstanding comprehensive traits.

In terms of yield traits, there was no significant difference between ZUPM01 and 9311 in terms of plant height, effective panicle, panicle length, total grain number, solid grain number, seed setting rate, yield per plant, dry matter weight, and thousand-grain weight (p>0.05) (Table 2) .

Table 2 Investigation results of ZUPM01 yield and test traits

Values are expressed as the mean ± standard deviation of three biological repetitions, and the significance of the differences in each trait among different materials was analyzed by LSD method (α = 0.05).

Example 4 The flanking sequence of the exogenous sequence of the transformation event ZUPM01 and the insertion position in the rice genome

Primers (SEQ ID NO: 16-SEQ ID NO: 29) for flanking sequence separation were designed, and the right flanking sequence (SEQ ID NO: 5, 6867-7306) of the transformant ZUPM01 was obtained by FPNI-PCR amplification and sequencing. position), and the left flank sequence (position 1-486 of SEQ ID NO: 5) was obtained by PCR amplification and sequencing according to the genome sequence. In the PlantGDB database (http://www.plantgdb.org/OsGDB/cgi-bin/blastGDB.pl), the BLASTN tool was used to perform a homologous comparison analysis of the flanking sequences and the rice genome sequence, with MSU 7 as the reference sequence. Analysis showed that the transformant ZUPM01 was inserted into the rice genome at Chr06: 2736689-2736704.

The operation steps of FPNI-PCR are as follows:

1) Extract rice genomic DNA.

2) With the genomic DNA in step 1) as the template of the first round of PCR reaction, the reaction system is as follows:

The reaction procedure is:

95°C, 2.5min; (94°C, 10sec; 62°C, 30sec; 72°C, 2min) × 2 cycles;

94℃, 10sec;

25℃, 2min;

72°C (5.1% ramp), 2min;

[94°C, 10sec; 62°C, 30sec; 72°C, 2min; 94°C, 10sec; 62°C, 30sec; 72°C, 2min; 94°C, 10sec; 44°C, 30sec; 72°C, 2min;]

×5 cycles;

72°C, 5min;

25°C, 10min.

3) Using the corresponding first-round PCR product in step 2) as a template to perform a second-round PCR amplification.

The reaction system is as follows:

The reaction procedure is:

95℃, 1min 30sec;

[94°C, 10sec; 62°C, 30sec; 72°C, 2min] × 30 cycles;

72°C, 7min;

25°C, 10min.

4) Using the corresponding second-round PCR product (diluted 50 times) in step 3) as a template to perform the third-round PCR amplification.

The reaction system is as follows:

The reaction procedure is:

95℃, 1min 30sec;

[94°C, 10sec; 62°C, 30sec; 72°C, 2min] × 30 cycles;

72°C, 7min;

25°C, 10min.

5) The product of the third round of PCR was detected by electrophoresis in 1% (w/v) 1×TAE agarose gel, and DNA fragments above 250 bp were recovered.

6) Ligate the recovered fragments to the T vector, and ligate overnight at 16°C.

7) Transform the ligation product in 6).

8) Pick the positive clones and shake the bacteria to extract the plasmids, and send the plasmids for sequencing.

9) The sequencing results were compared with the rice genome sequence using the BLASTN tool in the PlantGDB database and the MSU database as the reference genome, and the best matching result was used as the insertion site.

The full-length insert sequence of ZUPM01 was further amplified and sequenced by overlapping PCR fragmentation. The actual insertion sequence of the transformant ZUPM01 and the rice genome sequence of the left and right flanks are shown in SEQ ID NO:5.

Table 3 Primer information used for flanking sequence isolation

1: The unit is bp.

Example 5 Detection of copy number of transformation event ZUPM01

The copy number of foreign gene insertion was determined by Southern blot hybridization.

Insertion copy number hybridization detection of OsPHF1 gene fragment Select four restriction endonucleases EcoRI, PvuII, NheI and HindIII to digest positive control pPHF1G9A-1300 plasmid, negative control wild-type 9311 genomic DNA and different generations (T ₅ and T ₆ ) ZUPM01 transformant genomic DNA. After running the gel and transferring to the membrane, it was labeled with the OsPHF1 gene probe (SEQ ID NO: 30). The hybridization results are shown in Figure 3A. The probe position of the target gene OsPHF1 and the restriction endonucleases EcoRI, PvuII, NheI and HindIII are shown in Figure 3B.

The OsPHF1 gene is an endogenous rice gene. After EcoRI, PvuII, NheI and HindIII digestion of the genome of the negative control 9311, a band was obtained by hybridization with the labeled probe, of which EcoRI digestion can mark a 1.5 kb band (lane 4 ), PvuII digestion can mark a 2.3kb band (lane 10), NheI digestion can mark a 9.4kb band (swimming lanes 15, 16), HindIII digestion can mark a 15kb band (swimming lane 23). It can be seen that the OsPHF gene has one copy in the non-transgenic rice. Simultaneously, after the negative control wild-type 9311 was marked with EcoRI, PvuII, NheI and HindIII three restriction endonucleases, no bands other than the endogenous gene were seen. In the blank control (swimming lanes 2, 8, 14, 21 ) did not hybridize to a band, indicating the specificity of the hybridization probe.

EcoRI has only one restriction site in the T-DNA region, which is located on the left side of the OsPHF1 gene probe. The labeled band obtained after the enzyme-digested ZUPM01 transformant genomic DNA is hybridized with the specific probe should include a 4.0kb T- The DNA sequence and the sequence of unknown size on the right genome, the entire fragment length is greater than 4.0kb. Two hybridization bands were marked in the experiment, one of which was a 1.5 kb band marked by the endogenous OsPHF1 gene, and the size of the band marked by the exogenous OsPHF1 gene was about 9.4 kb (lane 5, 6), which was in line with expectations. Meanwhile, EcoRI has no restriction site in the backbone region of the vector, so the size of the band marked by the positive control is the same as that of the plasmid, which is 12.7 kb (lane 3).

PvuII also has only one restriction site in the T-DNA region, which is located on the right side of the OsPHF1 gene probe. The labeled band obtained after the enzyme-digested ZUPM01 transformant genomic DNA hybridizes with the specific probe should include a 6.4kb The T-DNA sequence and the sequence of unknown size on the left genome, the entire fragment length is greater than 6.4kb. Two hybridization bands were marked in the experiment, one of which was a 2.3 kb band marked by the endogenous OsPHF1 gene, and the size of the band marked by the exogenous OsPHF1 gene was about 9.3 kb (lanes 11 and 12), which was in line with expectations. At the same time, PvuII has no restriction site in the backbone region of the vector, so the size of the band marked by the positive control is the same as that of the plasmid, which is 12.7 kb (lane 9).

NheI has only one restriction site in the T-DNA region, which is located on the left side of the OsPHF1 gene probe. The labeled band obtained after the enzyme-digested ZUPM01 genomic DNA is hybridized with the specific probe should include 3.7kb of T-DNA Sequences and sequences of unknown size on the genome to the right, the entire fragment is greater than 3.7kb in length. Two hybridization bands were marked in the experiment, one of which was a 9.4 kb band marked by the endogenous OsPHF1 gene, and the size of the band marked by the exogenous OsPHF1 gene was about 4.3 kb (lanes 17 and 18), which was in line with expectations.

HindIII has two restriction sites in the T-DNA region, which are located on both sides of the OsPHF1 gene probe, and will cut the T-DNA region into a 2.3kb fixed-size fragment. Two hybridization bands were marked in the experiment, one of which was the 15kb band marked by the endogenous OsPHF1 gene, and the band size marked by the exogenous OsPHF1 gene was 2.3kb (lane 24, 25), compared with the positive control plasmid enzyme The bands labeled with cleavage have the same size (lane 22), indicating that the probe-labeled band is indeed on the T-DNA region, and there is no recombination within the OsPHF1 gene.

Inserted copy number hybridization detection of G9A gene fragments NheI, HindIII and SacI three restriction endonucleases were selected to digest the blank control, positive control pPHF1G9A-1300 plasmid, negative control wild-type 9311 genomic DNA and different generations of ZUPM01 transformant genomic DNA . Labeled with G9A gene probe (SEQ ID NO: 31) after running the gel and transferring to the membrane. The hybridization results are shown in Figure 4A. The probe position of the target gene G9A and the restriction endonucleases NheI, HindIII and SacI are shown in Figure 4B.

NheI has a restriction site in the T-DNA region, which is located on the right side of the G9A gene probe. The labeled band obtained after the enzyme-digested ZUPM01 transformant genomic DNA is hybridized with the specific probe should include a 2.7kb T - DNA sequence and sequence of unknown size on the left genome, the entire fragment length is greater than 2.7kb. A single hybridization band was marked in the experiment, with a size of about 4.3 kb (lanes 5 and 6), which was in line with expectations. At the same time, Nhel has a restriction site in the backbone region of the vector, which is about 3.2 kb away from the left border, so the size of the band marked by the positive control is 6.0 kb (lane 3).

HindIII has two enzyme cutting sites in the T-DNA region, both of which are located on the right side of the G9A gene probe. The labeled band obtained after the enzyme-digested ZUPM01 transformant genomic DNA hybridizes with the specific probe should include 3.8kb The T-DNA sequence and the sequence of unknown size on the left genome, the entire fragment length is greater than 3.8kb. A single hybridization band was marked in the experiment, with a size of about 7.0 kb (lanes 11 and 12), which was in line with expectations. At the same time, HindIII has no restriction site in the backbone region of the vector, so the size of the band marked by the positive control is 10.4 kb (lane 9).

SacI has four enzyme cutting sites in the T-DNA region, and the sites on both sides of the G9A gene probe will cut the T-DNA region into a 4.6kb fixed-size fragment. In the experiment, a single hybridization band was marked about 4.6kb (lane 17, 18), which was the same size as the band marked with positive control plasmid digestion (lane 15), indicating that the probe-labeled band was indeed in the T-DNA region, and no recombination occurred within the G9A gene. Simultaneously, after the negative control wild-type 9311 was marked with three restriction endonucleases NheI, HindIII and SacI, no bands were seen (lanes 4, 10, 16), and no bands were seen in the blank control (lanes 2 , 8, 14), indicating the specificity of the hybridization probe.

The above experimental results indicated that the ZUPM01 transformant contained a single copy of exogenous OsPHF1 and G9A gene fragments.

Example 6 The tolerance of the transformation event ZUPM01 to glyphosate herbicides

In a field environment that meets the isolation requirements, the tolerance of the transformants to the herbicide glyphosate was determined by artificial spraying of herbicides, and the effectiveness of the target traits of herbicide tolerance of the transformants was clarified. The experiment was repeated three times, each plot was 1m ² (1m×1m), and the interval between plots was 0.5m. The experimental treatments were set without spraying herbicides on transgenic rice, spraying target herbicide on transgenic rice, not spraying herbicide on corresponding non-transgenic rice, and spraying target herbicide on corresponding non-transgenic rice. The spraying concentration of herbicide is 1 times (1×, 200mL/mu or 82g/mu) and 2 times (2×, 400mL/mu or 164g/mu) of spraying clear water (0×) and the recommended dose in the field respectively. and 4 times (4×, 800mL/mu or 328g/mu).

After the target herbicide glyphosate was sprayed, the number of normal seedlings was investigated 1 week and 2 weeks after the application, and the number of normal seedlings and phytotoxic seedlings (grade 2-5) were investigated 4 weeks after the application. One week after treatment, the plants of 9311 and the transformants sprayed with clean water could survive normally, but overgrown with weeds; most of the plants of the recipient control 9311 sprayed with glyphosate died, even if a small part of them did not completely lose chlorosis The heart leaves were also dead; while the transformant ZUPM01 sprayed with different doses of glyphosate could grow normally. After 2 weeks and 4 weeks after the application of the drug, the weeds of the 9311 and the transformant plants sprayed with clear water were more luxuriant, which seriously affected the normal growth of rice; the recipient control 9311 sprayed with glyphosate had all dried up and died; The transformant ZUPM01 treated with different doses of glyphosate can still grow normally.

The results of the investigation on the phytotoxicity of the transformant ZUPM01 at 1 week, 2 weeks, and 4 weeks after the application showed that no phytotoxicity occurred under the treatments of various doses of herbicides, and the phytotoxicity rate was 0%, and no grade 4 and 5 level phytotoxicity seedlings (Table 4). According to the criteria for determining the tolerance level of rice to herbicides, it can be judged that the tolerance level of the transformant ZUPM01 to the target herbicide glyphosate is excellent.

Table 4 The tolerance performance of ZUPM01 to glyphosate

Doses are expressed in multiples of the recommended dosage of herbicides in the field. Values are expressed as the mean ± standard deviation of three biological repetitions, and the statistical analysis is tested by the LSD method.

The results showed that the recipient control 9311 could not tolerate the medium dose of glyphosate at the recommended dose, while the transformant ZUPM01 could tolerate at least 4 times the medium dose of glyphosate at the recommended dose, and the tolerance to the target herbicide reached "excellent". Level, with good promotion value.

Example 7 Tolerance of transformation event ZUPM01 to soil low phosphorus stress

Under natural conditions in the field, 4 treatments were set up, divided into no-phosphorus, low-phosphorus, medium-phosphorus, and high-phosphorus. The field plots that almost did not contain phosphorus elements treated by our laboratory for many years were used for experiments. The fertilization scheme of each treatment was as follows:

Phosphorus-free treatment (NP): superphosphate 0kg/mu, urea 30kg/mu, potassium chloride 20kg/mu;

Low phosphorus treatment (LP): superphosphate 7.5kg/mu, urea 30kg/mu, potassium chloride 20kg/mu;

Medium phosphorus treatment (MP): superphosphate 15kg/mu, urea 30kg/mu, potassium chloride 20kg/mu;

High phosphorus treatment (HP): superphosphate 30kg/mu, urea 30kg/mu, potassium chloride 20kg/mu.

The dry matter weight and phosphorus content of the stems and leaves were measured at the full tillering stage, the booting stage and the mature stage (late wax ripening stage), and the dry matter weight and phosphorus content of the flag leaves, ear stalks, chaff and brown rice were measured at the mature stage, and Calculate the cumulative amount of phosphorus according to the dry matter weight and phosphorus content.

Phosphorus nutrient efficiency consists of three parts: absorption efficiency, utilization efficiency and transport efficiency. This project uses the accumulation of phosphorus in the aboveground part of the plant to measure the phosphorus uptake efficiency of the plant; the dry matter produced by the unit phosphorus uptake is defined as the phosphorus use efficiency in the plant; the percentage of phosphorus in the reproductive part of the total phosphorus in the shoot after harvest is the phosphorus transfer efficiency.

In the mature stage, the harvested plots were harvested and weighed in the sun, and converted into yield per mu.

After carrying out the phosphate fertilizer treatment, the total phosphorus, organic matter, available phosphorus and pH value of the test plot were detected, and the results showed that the total phosphorus and available phosphorus contents in the treatments of no phosphorus, low phosphorus, medium phosphorus and high phosphorus showed a significant upward trend (Table 5 ), indicating that the experimental treatment was effective and in line with expectations.

Table 5 Physical and chemical properties of soil

1: The unit is "mg/kg", and the result comes from the mean of 3 repetitions.

The dry matter weight and phosphorus content of flag leaves, ear stalks, chaff and brown rice were measured at the mature stage, and the cumulative amount of phosphorus was calculated according to the dry matter weight and phosphorus content, and the efficiency of phosphorus absorption, utilization and transport was calculated. The results are shown in Table 6.

Table 6 Phosphorus absorption, utilization and transport efficiency of ZUPM01

Values are expressed as the mean ± standard deviation of three biological repetitions, and the statistical analysis is tested by the LSD method (α = 0.05). The letter before "/" indicates the significance of the data difference among different treatments, and the letter after "/" indicates the significance of the data difference between different materials. "NP, LP, MP, HP" represent no phosphorus treatment, low phosphorus treatment, medium phosphorus treatment and high phosphorus treatment, respectively.

The phosphorus uptake, utilization and transport efficiency of the acceptor 9311 increased with the increase of soil phosphorus level, while the phosphorus uptake, utilization and transport efficiency of the transformant ZUPM01 had no significant difference under different phosphorus concentration treatment conditions. The phosphorus uptake, utilization and transport efficiency of ZUMP01 was significantly higher than that of acceptor 9311 under no and low phosphorus treatments, while the phosphorus uptake, utilization and transport efficiency of ZUMP01 and acceptor 9311 had no significant difference under medium and high phosphorus treatments sexual difference.

Investigated the yield under 4 phosphorus concentration treatments, the result is as shown in table 7, the yield of contrast 9311 increases continuously along with the rising of soil phosphorus concentration, has shown its sensitivity to phosphorus treatment; There was no significant difference in yield under the treatments, and the tolerance to phosphorus deficiency stress was stronger. The yields of ZUPM01 transformants were 597.5±31.8 and 609.7±35.6kg/mu under no-phosphorus and low-phosphorus treatments, which were significantly higher than those of 9311 (485.4±47.71 and 497.6±56.2kg/mu). The yields under the treatments were 719.1±189.1kg/mu and 633.1±125.5kg/mu, respectively, which had no significant difference from the control.

Table 7 Yield performance of transformants under different phosphorus concentration treatments (kg/mu)

Values are expressed as the mean ± standard deviation of three biological repetitions, and the statistical analysis is tested by the LSD method (α = 0.05). The letter before "/" indicates the significance of the data difference under different treatments, and the letter after "/" indicates the significance of the data difference between different materials. "NP, LP, MP, HP" stand for no-phosphorus, low-phosphorus, medium-phosphorus and high-phosphorus treatments, respectively.

The above results show that the expression of the target gene OsPHF1 in the transformant ZUPM01 can enhance the phosphorus nutrient efficiency of rice under the conditions of phosphorus-free and low-phosphorus stress, thereby improving the physiological traits of rice and increasing rice yield.

Example 8 Detection method of transformation event ZUPM01

Such as agricultural products or commodities can be produced from the transgenic rice event ZUPM01. If a sufficient expression level is detected in the agricultural product or commodity, the agricultural product or commodity is expected to contain a nucleotide sequence capable of diagnosing the presence of the transgenic rice event ZUPM01 material in the agricultural product or commodity. Such agricultural products or commodities include, but are not limited to, rice flour, rice oil, rice bran, rice germ, rice protein, rice starch, rice bran nutritive oil or rice bran polysaccharide and any other food that is to be consumed by animals as a food source, or otherwise as a bulking agent or The ingredients in the cosmetic composition are used for cosmetic purposes and the like. Nucleic acid detection methods and/or kits based on probes or primer pairs can be developed to detect the transgenic rice event ZUPM01 nucleotide sequence such as SEQ ID NO: 1 or SEQ ID NO: 2 shown in biological samples, wherein the probe sequence Or the primer sequence is selected from the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 , to diagnose the presence of transgenic rice event ZUPM01.

One of the detection methods is: use the PCR method to detect the specific border sequences in the plants of two generations (T ₅ and T ₆ ) of ZUPM01, and the PCR primer pairs used are SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 9 and SEQ ID NO: ID NO: 10, SEQ ID NO: 11, PCR reaction system:

The reaction procedure is:

94°C, 5min; (94°C, 30sec; 55°C, 30sec; 72°C, 1.0min) × 35 cycles; 72°C, 7min; 4°C, 5min.

The PCR product was detected by electrophoresis in 1% (w/v) 1×TAE agarose gel, and the results are shown in FIG. 5 . The expected target band can be amplified in the transformation event, but the target band cannot be amplified in other samples not derived from the transformation event.

In summary, the transgenic rice event ZUPM01 of the present invention has high tolerance to glyphosate herbicide and low phosphorus stress, and the detection method can accurately and quickly identify whether the DNA molecule of the transgenic rice event ZUPM01 is contained in the biological sample.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be The scheme shall be modified or equivalently replaced without departing from the spirit and scope of the technical scheme of the present invention.

Claims (24)

1. a kind of nucleic acid sequence, which is characterized in that the nucleic acid sequence include SEQ ID NO:1 or its complementary series, and/or SEQ ID NO:2 or its complementary series, the nucleic acid sequence are originated from transgenic paddy rice event ZUPM01.

2. nucleic acid sequence according to claim 1, which is characterized in that the nucleic acid sequence include SEQ ID NO:3 or its Complementary series, and/or SEQ ID NO:4 or its complementary series.

3. nucleic acid sequence according to claim 2, which is characterized in that the nucleic acid sequence include SEQ ID NO:5 or its Complementary series.

4. a kind of method existing for DNA of test sample transgenic rice event ZUPM01 characterized by comprising

Contact sample to be tested in nucleic acid amplification reaction at least two primers；

Carry out nucleic acid amplification reaction；

Detect the presence of amplified production；

The amplified production includes SEQ ID NO:1 or its complementary series, and/or SEQ ID NO:2 or its complementary series；

The amplified production is originated from transgenic paddy rice event ZUPM01.

5. method existing for the DNA of test sample transgenic rice event ZUPM01, feature exist according to claim 4 In the amplified production further includes SEQ ID NO:6 or its complementary series, and/or SEQ ID NO:7 or its complementary series.

6. method existing for the DNA of test sample transgenic rice event ZUPM01 according to claim 4 or 5, special Sign is that the primer includes the first primer and the second primer, and the first primer is selected from SEQ ID NO:1 or its complementary sequence Column, SEQ ID NO:8 and SEQ ID NO:10；Second primer is selected from SEQ ID NO:2 or its complementary series, SEQ ID NO:9 and SEQ ID NO:11.

7. a kind of method existing for DNA of test sample transgenic rice event ZUPM01 characterized by comprising

Contact sample to be tested with probe, the probe includes SEQ ID NO:1 or its complementary series or SEQ ID NO: 2 or its complementary series, the source probe transgenic rice event ZUPM01；

Hybridize the sample to be tested and the probe under stringent hybridization conditions；

Detect the hybridisation events of the sample to be tested and the probe.

8. method existing for the DNA of test sample transgenic rice event ZUPM01, feature exist according to claim 7 In the probe further includes SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.

9. special according to method existing for the DNA of the test sample transgenic rice event ZUPM01 of claim 7 or 8 Sign is that at least one described probe is marked at least one fluorophor.

10. a kind of method existing for DNA of test sample transgenic rice event ZUPM01 characterized by comprising

Contact sample to be tested with marker nucleic acid molecules, the marker nucleic acid molecules include SEQ ID NO:1 or it is mutual Complementary series or SEQ ID NO:2 or its complementary series, the marker nucleic acid molecules are originated from transgenic paddy rice event ZUPM01；

Hybridize the sample to be tested and the marker nucleic acid molecules under stringent hybridization conditions；

The hybridisation events of the sample to be tested and the marker nucleic acid molecules are detected, and then pass through marker assistant breeding point Analysis is to determine that herbicide tolerant and/or low-phosphorus stress tolerance and marker nucleic acid molecules are chain on science of heredity.

11. method existing for the DNA of test sample transgenic rice event ZUPM01 according to claim 10, feature It is, the marker nucleic acid molecules further include SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or it is complementary Sequence.

12. a kind of DNA detection kit, which is characterized in that including at least one DNA molecular, the DNA molecular includes SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series, can be used as transgenic paddy rice event ZUPM01 or its offspring have one of DNA primer of specificity or probe；The DNA molecular is originated from transgenic paddy rice event ZUPM01。

13. DNA detection kit according to claim 12, which is characterized in that further include when the DNA molecular is as probe SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.

14. a kind of method for protecting rice plants from the damage as caused by herbicide, which is characterized in that including that will contain effectively Dosage glyphosate herbicidal is applied to the big Tanaka for planting at least one transgenic rice plant, and the transgenic rice plant exists Successively comprising SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 in its genome, or It include SEQ ID NO:5 in the genome of transgenic rice plant described in person；The transgenic rice plant has to glyphosate The tolerance of herbicide.

15. a kind of method for protecting rice plants from the damage as caused by soil low-phosphorous element, which is characterized in that be included in low At least one transgenic rice plant is planted in phosphorus concentration soil, the transgenic rice plant successively includes in its genome SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 or the transgenic paddy rice are planted It include SEQ ID NO:5 in the genome of object；The transgenic rice plant has the tolerance to low-phosphorus stress.

16. a kind of method for the big Tanaka weeds for controlling rice cultivation plant, which is characterized in that including effective dose grass will be contained Sweet phosphine herbicide is applied to the big Tanaka for planting at least one transgenic rice plant, and the transgenic rice plant is in its gene It successively include SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 or described in group It include SEQ ID NO:5 in the genome of transgenic rice plant；The transgenic rice plant has to glyphosate herbicidal Tolerance.

17. a kind of method that culture has the rice plants of tolerance to glyphosate herbicidal characterized by comprising

An at least rice paddy seed is planted, includes the nucleic acid sequence of specific region, the spy in the genome of the rice paddy seed The nucleic acid sequence for determining region successively includes SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID The nucleic acid sequence of NO:2 or the specific region includes SEQ ID NO:5；

The rice paddy seed is set to grow up to rice plant；

The rice plant is sprayed with effective dose glyphosate herbicidal, harvest does not have the nucleic acid of the specific region with other The plant of sequence compares the plant with the plant injury weakened.

18. a kind of method that culture has the rice plants of tolerance to low-phosphorus stress characterized by comprising

An at least rice paddy seed is planted in low-phosphorous soil, includes the nucleic acid of specific region in the genome of the rice paddy seed Sequence, the nucleic acid sequence of the specific region successively include 561-6647 SEQ ID NO:1, SEQ ID NO:5 nucleic acid sequences The nucleic acid sequence of column and SEQ ID NO:2 or the specific region includes SEQ ID NO:5；

The rice paddy seed is set to grow up to rice plant；

Harvest the plant compared with the plant of other nucleic acid sequences for not having the specific region with the plant injury weakened.

19. a kind of method for generating the rice plant that there is tolerance to glyphosate herbicidal, which is characterized in that including to described 561-6647 nucleic acid sequences of SEQ ID NO:5 are introduced in the genome of rice plant, and make the base of the rice plant Include successively SEQ ID NO:1,56I-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 because organizing, or makes The genome of the rice plant includes SEQ ID NO:5, the rice plant of selection tolerance glyphosate.

20. 9 method for generating the rice plant that there is tolerance to glyphosate herbicidal according to claim 1, feature It is, comprising:

By transgenic paddy rice event ZUPM01 the first parent rice plant to glyphosate herbicidal with tolerance and lack grass Second parent's rice plant sexual hybridization of sweet phosphine tolerance, to generate a large amount of progeny plants；

The progeny plant is handled with glyphosate herbicidal；

The progeny plant of selection tolerance glyphosate.

21. a kind of method for generating the rice plant that there is tolerance to low-phosphorus stress, which is characterized in that including to the rice 561-6647 nucleic acid sequences of SEQ ID NO:5 are introduced in the genome of plant, and make the genome of the rice plant Successively include SEQ ID NO:1,561-6647 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2, or makes described The genome of rice plant includes SEQ ID NO:5, the rice plant of selection tolerance low-phosphorus stress.

22. the generation according to claim 21 has the method for the rice plant of tolerance to low-phosphorus stress, which is characterized in that Include:

By transgenic paddy rice event ZUPM01 the first parent rice plant to low-phosphorus stress with tolerance and lack the low-phosphorous side of body The second parent's rice plant sexual hybridization for compeling tolerance, to generate a large amount of progeny plants；

The progeny plant is handled with low-phosphorus stress；

The progeny plant of selection tolerance low-phosphorus stress.

23. a kind of composition for being produced from transgenic paddy rice event ZUPM01, which is characterized in that the composition is rice, rice Grass, rice husk or seed rice.

24. a kind of agricultural product or commodity produced by transgenic paddy rice event ZUPM01, which is characterized in that the agricultural product or quotient Product are rice flour, rice bran oil, rice bran, rice embryo, rice gluten, rice starch, Rice Bran oil or rice bran polysaccharide, cosmetics or filler.